Methods for the isolation, recovery and purification of non-polar compounds using novel hydrophobically-modified polysaccharide gels

ABSTRACT

A process is described for the preparation f electrostatically-linked, aliphatic- or alicyclic-substituted anionic or cationic polysaccharide gels from readily available macroporous ionic polysaccharide chromatographic media, such as diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and sulfopropyl (SP) substituted polysaccharide gels. These novel gels are used for the isolation, recovery and purification of non-polar extractives using one or more extracting solvents from the group of lower alcohols, ketones, and water. The non-polar extractives may be alk(n)lyresorcinols, steroid, triterpenoid, cardiac glycosides and saponins, steryl ferulates and other phenolic acid conjugates, flavonoids, lipids, alcohol-soluble antimicrobials, prolamines or other alcohol-soluble proteolipid complexes.

This application is a divisional of U.S. application Ser. No. 09/613,110now U.S. Pat. No. 6,582,594 filed on Jul. 10, 2000. U.S. applicationSer. No. 09/613,110 is a continuation of copending PCT applicationCA99/00004, filed Jan. 11, 1999, which designated the United States andclaimed the priority of U.S. application Ser. No. 60/071,251 filed Jan.12, 1998. The priorities of all are claimed herein, and the entiredisclosures of all are incorporated herein by reference.

The present invention relates to a process for the preparation ofelectrostatically-linked, aliphatic- or alicyclic-substituted anionic orcationic polysaccharide gels from readily available macroporous ionicpolysaccharide chromatographic media. The present invention furtherrelates to the isolation, recovery and purification of non-polarextractives using said polysaccharide gels in a process of hydrophobicinteraction chromatography, for the absorption and desorption of theextractives in the presence of and as a result of the concentration andselection of an organic solvent.

BACKGROUND OF THE INVENTION

Agricultural plants, and waste streams from their processing, by way ofan example, may contain components that are now being discovered ashaving desirable therapeutic and other benefits. For example: somesaponins have been shown to exhibit antineoplastic chemotherapeuticvalue (U.S. Pat. No. 5,558,866), while others find use in the treatmentof hypercholesterolemia (U.S. Pat. No. 5,502,038). Still furtherantifungal (e.g. Crombie, W. M. L., and Crombie, L., Phytochemistry 25:2069-2073, 1986) and immunogenic (U.S. Pat. No. 5,597,807) activitiesare known as well as surfactant, emulsifying and foam stabilizingproperties which are summarized by Price et al. (CRC Crit. Rev. FoodSci. Nutr., 26, 27-135, 1987). These are but a few examples from theliterature.

In addition, some flavones and their glycosides are known to exhibitantimutagenicity (e.g. Peryt, B., et al., Mutation Res. 269: 201-215,1992), and antitumor activity (e.g. Wei, H., et al., Cancer Res. 50:499-502, 1990). Further reports of beneficial biological activities andfunctional properties can be found in a number of reviews (e.g. “Plantflavonoids in biology and medicine II. Biochemical, cellular, andmedicinal properties” Ed. By V. Cody, E. Middleton Jr., J. B. Harborne,and A. Beretz, Liss Inc, New York, 1988.)

Canadian Patent Applications 2,006,957 and 2,013,190 describeion-exchange processes carried out in aqueous ethanol to recover smallquantities of high value byproducts from cereal grain processing waste.According to CA 2,013,190, an alcoholic extract from a cereal grain isprocessed through either an anionic and/or cationic ion-exchange columnto obtain minor but economically valuable products. The anionic,cationic and neutral fractions were analysed by thin-layerchromatography and a number of components were identified. For examplein an anionic fraction from an alcoholic extraction of hull-less wholeoats, the following components were identified: phenolic acids,including ferulic acid, p-coumaric acid and caffeic acid; alkaloids suchas avenanthramides; fatty acids, organic acids and amino acids. From thesame alcoholic extract the neutral fraction contained compounds, suchas: free sugars; phenolics, such as flavonoids; saponins such asavenacosides and desglucosyl-avenacosides; alkaloids such as theavenacins; and various polar lipids. The compounds identified in thevarious fractions were not individually isolated by ion-exchangechromatography since many carried the same net charge under theconditions used and thus, this method alone is of little value in theisolation of these useful components for industrial or commercial use.Furthermore, the extractives to be isolated in the present invention arefor the most part neutral under conditions used, and thus cannot beisolated by ion exchange chromatography alone, which sorts moleculesaccording to charge.

PCT application WO 92/06710, discloses both the composition andisolation/separation technologies of Quillaja saponins for end uses asimmunogenic complexes, using repeated semi-preparative high performanceliquid chromatography (HPLC) on a reverse-phase column with anacetonitrile:water gradient elution. The scale of the separation appearsnot to be intended for production of significant quantities forcommercialization but rather for proof of efficacy. The isolatedproducts were produced only on the microgram scale. The scale-up of theseparation technique for commercial applications was not disclosed.

U.S. Pat. No. 5,094,960 describes methods of removal of processchemicals from labile biological mixtures by hydrophobic interactionchromatography (HIC) using a resin comprising octadecyl chains coupledto a silica matrix. A method of removing lipid soluble process chemicalssuch as synthetic detergents, chemical inducers and organic solventsfrom aqueous biological fluids, particularly directed to producing aprotein-containing composition such as blood plasma, cryoprecipitates,and blood plasma fractions, was described. In this disclosure materialsand conditions are employed that minimize adsorption and separation ofproteins and maximize the removal of the process chemicals.Substantially no biological material is retained on the column.Furthermore, no indication is given as to the intended field of use ofany of the compounds and chemicals adsorbed in the process, nor specificconditions to selectively recover any of the adsorbed componentsretained on the column.

A number of different procedures are known for the isolation andpurification of isoflavones. D. E. Walter described a procedure for thepreparation of the isoflavone genistin and its aglycone genistein fromdefatted soybean flakes (J. Amer. Chem. Soc. 63, 3273-3276, 1941). Theprocedure involved methanol extraction, acetone precipitation,centrifugations and several recrystallizations and gave only oneisoflavone, genistin, from which the aglycone genistein could preparedby acid hydrolysis. Ohta et al. described a procedure for isoflavoneextraction from defatted soybeans wherein the flakes were extracted withethanol and the ethanolic extract treated with acetone and ethylacetate. Column chromatography of the ethyl acetate fraction on silicagel and Sephadex LH-20™ in several additional solvents produced a numberof fractions from which individual isoflavones could be recovered byrepeated recrystallization (Agric. Biol. Chem. 43: 1415-1419, 1979).Essentially the same separation protocols were used by Farmakalidis, E.and Murphy, P. A. to separate isoflavones extracted using acidifiedacetone rather than ethanol (J. Agric. Food Chem. 33: 385-389, 1985).These publications are but a few of the many examples in the literaturefor the laboratory scale extraction and purifications of specificisoflavones. However due to issues of solvent handling and disposal aswell as economic feasibility, these procedures are hard to scale up to acommercial process and produce single compounds in undisclosed yields.

U.S. Pat. No. 5,679,806 addressed some of these issues, disclosing aprocess for the isolation and purification of isoflavones from plantmaterial. The process consisted of three steps whereby the plantmaterial is extracted, the resulting extract fractionated on a reversephase low pressure polymethacrylate or C₁₈ chromatography column bygradient elution of the adsorbed isoflavones from the column, andfinally the resulting fractions containing specific isoflavones areeluted from the column. This process differs in several significant waysfrom the process described in the embodiments disclosed herein. First,the present process is not restricted to the isoflavone components butalso yields a saponin fraction substantially free of isoflavones as wellas the entire group of isoflavones which, if desired, can be furtherfractionated for individual components. Secondly, the present processdoes not rely on methacrylate or C₁₈-substituted reverse phase inorganicsupport matrices, which generally display much lower loading capacitiesand are harder to clean in place than polysaccharide-based gels.Thirdly, the flexibility of the present process allows that conditionsbe varied, either to capture the isoflavones by absorption or to allowthem to elute through the column leaving other non-isoflavone componentsstill absorbed, simply by varying the amount of water in an aqueousalcohol washing solution.

U.S. Pat. No. 5,482,914 teaches that agarose-based gels can besynthesized/modified for the binding of lipoproteins by covalentlylinking glycidyl ethers of polyoxyethylene detergents of the typeHO—(CH₂CH₂O)_(n)—O—R to give a modified gel matrix suitable for theremoval of lipoproteins from human and animal body fluids. Thistechnology refers only to the chemical processes for producing the geland makes no claims either for electrostatic binding of ligands such aswe describe, or for any examples of separation or recovery from plantmaterial.

Thus, there is still a need for processes, chromatographic proceduresand improved absorption media that are adaptable to a wide range ofcompounds in a commercially viable manner that provide highconcentrations of these compounds which can be subsequently recovered inhigh yield, purity and in unaltered form. There is also a need for aprocess in which the chromatographic media can be regenerated andre-used many times to reduce both waste disposal costs and replacementcosts. Furthermore, for commercial scale production of non-polarextractives it would also be advantageous to reduce the direct contactof solvents such as chlorinated hydrocarbons (e.g. chloroform,dichloromethane), nitriles (e.g. acetonitrile), aromatics (e.g. benzene,toluene), other potentially undesirable reagents (e.g. salts, mineralacids, bases), and chromatographic media contaminants (methacrylate-,divinylbenzene-, styrene-monomers, silica etc.) from direct contact withdesired products. To accomplish this latter objective and still achievethe necessary separations, it would be desirable to selectively alterthe chromatographic media to achieve the separation required and useonly one simple, acceptable solvent, rather than to use a singlechromatographic media and rely on a wide range of more unacceptablesolvents. It is to these ends that this technology is directed.

SUMMARY OF THE INVENTION

The present invention relates to a process for the preparation ofelectrostatically-linked, aliphatic- or alicyclic-substituted anionic orcationic polysaccharide gels from readily available macroporous ionicpolysaccharide chromatographic media.

The present invention also relates to the electrostatically-linked,aliphatic- or alicyclic-substituted anionic or cationic polysaccharidegels so produced.

The present invention further relates to the isolation, recovery andpurification of non-polar extractives using said polysaccharide gels ina process of hydrophobic interaction chromatography, for the absorptionand desorption of the extractives in the presence of and as a result ofthe concentration and selection of an organic solvent.

Thus, according to the present invention there is provided a method ofpreparing an electrostatically-linked, aliphatic oralicyclic-substituted anionic or cationic polysaccharide gel from amacroporous ionic polysaccharide chromatographic matrix comprising thesteps of:

-   -   attaching, by ion exchange, a hydrophobic ligand containing a        strongly ionizable functional group of opposite charge to that        of the said polysaccharide, so that a substantial amount of the        available ionic sites of the said polysaccharide are occupied by        the ligand to form a modified hydrophobic phase component.

Further according to the present invention there is provided anelectrostatically-linked, aliphatic- or alicyclic-substituted anionic orcationic polysaccharide gel comprising a hydrophobic ligandelectrostatically bonded to a macroporous ionic polysaccharide gelmatrix, so that a substantial amount of the available ionic sites of thesaid ionic polysaccharide gel matrix are occupied by the ligand to forma modified hydrophobic phase component, wherein the ligand contains astrongly ionizable functional group of opposite charge to that of thesaid ionic polysaccharide gel matrix.

This invention is also directed to a method of isolating a non-polarextractive comprising:

-   -   contacting said non-polar extractive in an aqueous organic        solvent solution with a gel matrix selected from the group of an        electrostatically-linked, aliphatic- or an alicyclic-substituted        anionic or cationic polysaccharide gel matrix;    -   washing said gel matrix with said aqueous organic solvent        solution;    -   washing said gel matrix with additional aqueous organic solvent        solution,    -   wherein the proportion of the solvent in said additional        solution is increased; and    -   recovering said extractive from an effluent stream.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows the extraction and group separation of oat saponins.

FIG. 2 shows the separation of avenacin-type and avenacoside-type oatsaponins.

FIG. 3 a shows the HPLC-ELSD profile of oat avenacin saponins and FIG. 3b shows the HPLC-ELSD profile of oat avenacoside saponins.

FIG. 4 a shows the HPLC-ELSD profile of Octyl Sepharose CL-4B™ purifiedAvenacin A-1; FIG. 4 b shows the mass spectrum of purified Avenacin A-1;and FIG. 4 c shows the structure of Avenacin A-1 from oat groats.

FIG. 5 shows the isolation and group separation of saponins from quinoa.

FIG. 6 a and FIG. 6 b show the HPLC-ELSD assignments for the quinoasaponins.

FIG. 7 a shows the TLC profile of quinoa enriched saponin fraction. FIG.7 b shows the TLC profile of the quinoa Group 1 saponin fraction andFIG. 7 c shows the TLC profile of the quinoa Group 2 saponin fraction.

FIG. 8 shows the purification and separation of quinoa Group 1 and 2saponins.

FIG. 9 shows the relationship between ethanol concentration and chainlength for various K′ values of Avenacin A-1 on SP Sephadex C-25™ alkyltrimethylammonium columns with different chain lengths.

FIG. 10 shows the relationship between ethanol concentration on K′values as in FIG. 9, only expressed in terms of chain length.

DESCRIPTION OF PREFERRED EMBODIMENT

The present invention relates to a process for the preparation ofelectrostatically-linked, aliphatic- or alicyclic-substituted anionic orcationic polysaccharide gels from readily available macroporous ionicpolysaccharide chromatographic media. The present invention furtherrelates to the isolation, recovery and purification of non-polarextractives using said polysaccharide gels in a process of hydrophobicinteraction chromatography, for the absorption and desorption of theextractives in the presence of and as a result of the concentration andselection of an organic solvent.

The fundamental principles of both hydrophobic interactionchromatography (HIC) and reverse phase chromatography (RPC) are similar:chromatographic separation of components of a mixture based on theirdifferential affinities between a non-polar or hydrophobic ligandattached to a stationary phase, and a mobile phase. In RPC, thestationary phase usually, but not always, consists of an inorganic,pelliculate or particulate, hydrophilic support, typically silica, ontowhich a specific ligand with a relatively high degree of hydrophobicityhas been introduced at a complete or extremely high substitution rate toeffectively replace, mask or resurface the hydrophilic stationary phase.The mobile phase usually, but not always, contains an organic modifier(i.e. organic solvent) typically methanol, acetonitrile, ortetrahydrofuran. In HIC, the stationary phase typically consists of anorganic, hydrophilic, macroporous support, typically a chemicallymodified polysaccharide or polyacrylamide, onto which a specific ligandwith a relatively low degree of hydrophobicity, has similarly, butusually to a much lower substitution rate, been introduced to modify butnot necessarily eliminate the hydrophilic properties of the support. Themobile phase usually, but not always, consists of water containing abuffer or salt solution of variable concentration.

RPC is now the most commonly used technique for high performance liquidchromatography (HPLC) separation of relatively stable low molecularweight organic compounds. However, separation of many biologicalmacromolecules (e.g. proteins, peptides and nucleic acids) by RPC hasfound only limited application, because the stronger interaction with ahighly hydrophobic stationary phase and the use of organic solvents aseluent constituents can be very detrimental to the native structure ofthe macromolecules. As a result of these interactions, most of themacromolecules are subjected to unfolding and denaturation with theconcomitant loss of some or all of their biological activity.

HIC has developed as a practical alternative to RPC for the separationand purification of biological macromolecules because both theabsorption and desorption processes can be carried out in an aqueousbuffer by simply varying the salt concentrations, conditions that aremore favorable to the retention of biological activity of themacromolecule. However, few applications of HIC for the separation oflow molecular weight, relatively stable organic compounds appear to havebeen made. In accordance with the present invention, by maintaining afunctional hydrophilic core in addition to the hydrophobic ligand inclose spacial proximity, the HIC stationary phase displays uniqueadvantages for the separation and purification of amphiphiles (i.e.compounds containing both hydrophilic and hydrophobic regions) notavailable in most if not all RPC stationary phase chromatographic media.Examples of such amphiphilic extractives, include but are not limited tosaponins, flavonoids and prolamines, which contain hydrophilicsubstituents (i.e. glycosidic residues) attached to a relativelynon-polar backbone. If these amphiphiles additionally show stabilitytowards certain organic solvents such as the lower alcohols and ketones,including ethanol, isopropanol and acetone, these solvents can be usedto recover the components bound to the gel (i.e. running the HIC systemin an “RPC mode”) by either gradient, sequential or batch recoverytechniques known by the person skilled in the art of chromatography.

Furthermore, according to the present invention we have observed thatsuch HIC gels possess the capacity to clarify aqueous dispersions andmicelles known to be formed when such amphiphiles (i.e. aqueoussolutions and dispersions of saponins, flavonoids and prolamines abovetheir critical micellar concentration) are produced artificially orencountered in the extraction, concentration and purification or othermanipulation of aqueous alcoholic preparations from agricultural plantsand co-processing streams.

The hydrophobic interaction chromatography polysaccharide gel of thepresent invention can consist of a cationically substitutedpolysaccharide gel matrix made from a neutral polysaccharide of thepolyanhydrogalactan or polydextran class such as crosslinked agarose,containing covalently-linked cationically-charged functional groups suchas tertiary amines (DEAE, PEI) or quaternary amines (QAE,Trimethylamine) such as DEAE Sephadex A-25™ (diethlaminoethylpolydextran), QAE Sephadex A-25™ (quaternary aminoethyl polydextran) andQ Sepharose™ (quaternary amine crosslinked agarose) (Amersham PharmaciaBiotech., Piscataway, N.J.). According to this embodiment of the presentinvention these gels are modified by a hydrophobic ligand. The ligand isselected from an anionically-linked alkyl substitution of the class ofanionic detergents including alk(en)yl sulfonates and sulphates,alk(en)yl phosphonates and phosphates, mono- anddi-alky(en)ylphosphatidic acids. Said ligand can also include ananionically-linked alicyclic substitution of the class of anionicdetergents including taurocholates and taurodeoxycholates or by mixedligands of the class of anionic detergents includingalk(en)yl-arylsulfonates such as dodecylbenzenesulfonic acid andalk(en)ylbenzenesulfonates. According to this aspect of the inventionthe alk(en)yl substitution contains from about 4 to about 18 carbons.

The hydrophobic interaction chromatography polysaccharide gel can alsoconsist of an anionically substituted polysaccharide gel matrix madefrom a neutral polysaccharide of the polyanhydrogalactan or polydextranclass such as cross-linked agarose, containing covalently linkedanionically charged functional groups such as alk(en)yl sulfonates andalk(en)yl phosphonates. Specific examples include sulfopropyl (SP) suchas SP Sephadex C-25™ (sulfopropyl polydextran) (Pharmacia, Piscataway,N.J.). S Sepharose™ (methyl sulfonate crosslinked agarose) is a furtherexample of the polysaccharide gel matrix according to this embodiment ofthe invention. According to this embodiment of the present inventionthese gels are modified by a hydrophobic ligand. In this embodiment thehydrophobic ligand is a cationically-linked alkyl substitution of theclass of cationic detergents including alk(en)yltrimethylammoniumhalides such as CETRIMIDE™ (Sigma Chemical Co., St. Louis, Mo.) andquaternary alk(en)ylammonium halides such as tetrabutylammonium bromide(Sigma Chemical Co., St. Louis, Mo.), quaternary alk(en)ylpyridiniumhalides such as hexadecylpyridinium bromide, or alk(en)ylmagnesiumhalides and similar Grignard reagents. According to this aspect of theinvention the alk(en)yl substitution contains from about 4 to about 18carbons.

Thus, the present invention involves the production of specific HIC gelsfrom readily available starting materials by simple ion exchangeprocedures as described herein. Basically, either of the two differenttypes of ion exchangers, anionic or cationic, preferably consisting ofan ionically-substituted polysaccharide gel, can be used as thehydrophilic core. To this core a hydrophobic ligand, chosen from theaforementioned classes of anionic or cationic detergents and containinga strongly ionizable functional group of opposite charge to that of thepolysaccharide, is attached by ion exchange so that all, orsubstantially all, of the available ionic sites in the originalpolysaccharide are occupied by the ligand to form a modified,hydrophobic phase component. By the term “all or substantially all” itis meant that at least 70% of the available ionic sites in the originalpolysaccharide are occupied by the ligand and can include up to at least90% of the available ionic sites.

As an example of the process of preparation of these novel gels, QAESephadex A-25™ anionic exchange chromatography gel, in the chloride formas received from the manufacturer and equilibrated in aqueous ethanol,for example aqueous 50% ethanol, was gravity packed into a column. Thesolvent need not necessarily be aqueous ethanol but any such solvent aswater, or aqueous methanol, isopropanol or acetone containing sufficientwater to effect swelling of the polysaccharide core matrix-would beeffective for the purpose of the present invention. The column is firstconverted to the hydroxide (i.e. OH⁻) form. In one example this step isaccomplished by passing an excess of hydroxide ion equivalents (for thequantity of gel being prepared) of a dilute base through the column andthen washing the column to neutral pH. In one example the dilute base isa 0.5N solution of NaOH in aqueous 50% ethanol and the washing solutionis also aqueous 50% ethanol. The base need not necessarily be NaOH butany base such as KOH, NH₄OH, etc. and, as outlined above, the solventneed not necessarily be aqueous 50% ethanol. The column is thenconverted to the desired novel anionic detergent substituted form bysimply passing excess ionic equivalents of that detergent through thecolumn and again washing the column to remove any excess detergent. Inone example the anionic detergent is 0.5N sodium dodecyl sulfate (SDS)in aqueous 50% ethanol and the washing solvent is also aqueous 50%ethanol. This procedure is essentially the same for any of theaforementioned anionic detergents, and would be familiar to any personskilled in the art of ion exchange chromatography.

As another example of the process for the preparation of these novelgels, SP Sephadex™ C-25 cationic exchange chromatography gel, in thesodium form as received from the manufacturer and equilibrated inaqueous ethanol, for example aqueous 50% ethanol, was gravity packedinto a column. The solvent need not necessarily be aqueous ethanol butany such solvent as water, or aqueous methanol, isopropanol or acetonecontaining sufficient water to effect swelling of the polysaccharidecore matrix. The column is first converted to the hydrogen (i.e. H⁺)form. In one example this step is accomplished by passing an excess ofhydrogen ion equivalents (for the quantity of gel being prepared) of adilute acid through the column and then washing the column to neutralpH. In one example the dilute acid is a 0.1N solution of HCl in aqueous50% ethanol and the washing solution is also aqueous 50% ethanol. Theacid need not necessarily be HCl but any acid such as H₂SO₄,trifluoracetic acid, H₃PO₄, etc. and, as outlined above, the solventneed not necessarily be aqueous 50% ethanol. The column is thenconverted to the desired novel cationic detergent substituted form bysimply passing excess ionic equivalents of that detergent through thecolumn and again washing the column to remove any excess detergent. Inone example the cationic detergent is 0.5N hexadecyltrimethylammonium(HDTMA⁺) bromide in aqueous 50% ethanol and the washing solvent is alsoaqueous 50% ethanol. This procedure is essentially the same for any ofthe aforementioned cationic detergents, and would be familiar to anyperson skilled in the art of ion exchange chromatography.

The use of these cationically substituted and anionically substitutedpolysaccharide gels opens a whole new area of selective hydrophobicinteraction separation technologies allowing the user to select anappropriate ionic counterion for attachment to the gel for specificapplications e.g. for the isolation of steroids one would usetaurocholic acid, or cholesterol sulfonate to bind estrogens, sexhormones etc.; one would use cis vs trans unsaturated alkyl groups toseparate isomer mixtures for health food ingredients, to mention only afew possibilities. A person skilled in the art of chromatography willreadily recognize other similar gels, which can be prepared for specificapplications.

As mentioned previously, the present invention also relates to the useof these gels for the isolation, recovery and purification of non-polarextractives. This aspect of the invention depends on the hydrophobicityof the non-polar extractives to be isolated and the change inhydrophobicity that results from altering the concentration of therecovery solvents, which can be achieved by adding more or less water tothe recovery solvent. More specifically, this embodiment of the presentinvention is based on the observed selective differences in thehydrophobic attraction between relatively non-polar extractivescontaining aliphatic and/or alicyclic functional groups, as compared tothose containing aromatic and/or olefinic functional groups, or neither;and an aliphatically- or alicyclically substituted polysaccharide-basedgel; and on the changes in this attraction that can be made by alteringthe composition of suitable solvents, simply by the addition of more orless water.

By non-polar extractives, it is meant that the extractives are onlypartially soluble in aqueous alcoholic solvents ranging in compositionfrom 5% to 95%. This term includes extractives that are relativelynon-polar. In one aspect of the present invention the groups ofcompounds that can be separated are non-polar compounds and are selectedfrom the group, but are not limited to: steroids and triterpenoidderivatives, such as saponins, cardiac glycosides and steryl conjugates;flavonoids, such as flavones, flavonols, isoflavones and all of theirglycosides; phenolic conjugates, such as aliphatic alcohol esters andamides; polar lipids, such as mono- and di-glycerides and theirderivates and alk(en)ylresorcinols; and prolamines, such as zein,avenin, hordein or gliadin. The non-polar compounds of the presentinvention include both naturally occurring compounds and syntheticcompounds.

By synthetic compounds it is meant any compound prepared by syntheticchemical means. The method of the present invention is particularlyuseful for the purification of synthetic compounds which havepharmaceutical or therapeutical value.

Naturally occurring compounds include the compounds referred to above,and also include compounds from algae, fungi and unicellular organisms.These naturally occurring compounds include compounds naturallyoccurring in the microorganism and also those produced by geneticallyaltered cells.

In one embodiment of the present invention the novel gels are used toisolate and purify saponins from different sources. Saponins aresapogenols (triterpenoid aglycones) containing up to five sugars.Saponins in general and soyasaponins in particular are gaining muchattention because of the growing market share of soybeans and also froma pharmacological standpoint. Soyasaponins have been reported to exhibithaemolytic, goitrogenic, antioxidative and hypolipidemic properties.They have also been shown to impart a bitter and astringent taste tosoy-based foods. Their isolation and characterisation is important forthe breeding of new varieties and for their production aspharmacologically active value-added products. However, because of theircomplex chemistry, known chemical isolation techniques (i.e.,saponification or solvent extraction) may yield low quantities orhydrolysed products.

In one example, the present invention was used for the isolation of oatsaponins. Two different types of saponins have been isolated from oatkernels: the avenacoside-type pentacyclic triterpenoid saponins and theavenacin-type steroidal saponins. Both types show selectiveantimicrobial activity (e.g. Wubben, J. P., et al., Phytopath. 86,986-992, 1996; Maizel, J. V., et al., Biochemistry 3, 424-426, 1964) andmay prove valuable as active ingredients in topical creams and lotionsfor health care products or as antimicrobials for agricultural seeddressing formulations.

In another embodiment of the present invention, the novel gels were usedfor the isolation of saponins from quinoa. Chenopodium quinoa Willd.(quinoa) is a grain native to South America. Much scientific interesthas been generated in recent years due to the grain's nutritional value(the protein content averaging 14% fresh weight) and its hardy growingcharacteristics. Unfortunately, the grain has had limited use as a humanfood source because of the bitterness of the seed coat, which is thoughtto be caused in part by the presence of saponins. These saponins areprimarily found in the outer layers of the grain including the perianthand pericarp. Traditionally, the grain has been washed with water toremove most of these bitter components, prior to consumption.

In the past decade, nearly two dozen saponins, both neutral and acidichave been isolated from quinoa (e.g. Mizui, F., et al., Chem. Pharm.Bull. 36: 1415-1418, 1988; Mizui, F., et al., Chem. Pharm. Bull. 38:375-377, 1990). These saponins contain one of three pentacyclictriterpene algycones; oleanolic acid, hederagenin or phytolaccagenicacids. The glycosidic moieties of the neutral saponins can consist ofany combination of glucose, galactose, or arabinose, producing mono, di,tri and tetraglycosides. The acidic saponins may contain acidicfunctionalities at C-28, or a glucuronic acid residue attached to C-3 ofthe triterpenoid backbone. In general, these saponins are fairly polarcontaining up to four glycosyl residues. Both diacids and bisdesmosideshave been isolated.

Recently, quinoa saponins have been used in pharmaceutical preparationsas immunological adjuvants (e.g. U.S. Pat. Nos. 5,597,807 and5,688,772). They have been shown to stimulate nonspecific immunity,enhance an immunological response to a selected antigen and enhancedmucosal absorption of an administered drug.

Methods to isolate saponins from quinoa have involved the extraction ofwhole seed or bran fractions by either water or methanol (e.g. Ridout,C., et al., J. Sci. Food Agric. 54: 165-176, 1991, and Meyer, B. N., etal., J. Agric. Food Chem. 38: 205-208, 990). In both procedures, theextracts are defatted by a non-polar solvent such as petroleum ether ordiethyl ether. Solvent partition (usually n-butanol/water) followed byclassic column chromatography using silica gel with methanol/chloroformhas resulted in the isolation of these compounds. These methods would bedifficult to commercially develop due to the use of harsh solvents,incomplete partitioning of components and variability in yields. Anumber of chemical, spectral, enzymatic and bioassay directed methods todetect quinoa saponins have been reported in the literature including GC(e.g. Burnouf-Radosevich, M., et al., Phytochemistry 24: 2063-2069,1984), HPLC (e.g., Ruales, J., and Nair, B., Food Chemistry 48: 137-143,1993), and TLC (e.g. Ng, K. G., et al., Food Chemistry 49: 311-315,1994). Individual saponins have been characterised by NMR and GC-MS(e.g. Ma, W-W., et al., J. Nat. Prod. 52, 1132-1135, 1989).

In one embodiment of the present invention, the compounds can beisolated from plant material. The term plant material includes productsof agriculture, viniculture, horticulture or aquaculture. Agriculturalplants include cereal grains, for example wheat, oats, rye, corn, rice,quinoa, amaranth, buckwheat, triticale or barley; or oilseeds, such assoybean, canola, flaxseed, sunflower, safflower or mustard; orpulsecrops, for example, peas, lentils or beans; or forage crops, suchas fescue, timothy, clover, alfalfa or wheatgrass; or herbs, such asparsley, rosemary, sage, or mint. The compounds can also be recoveredfrom their co-processing streams. However, the invention is not limitedto compounds isolated from plants or agriculture co-processing products.The invention can also be used to extract compounds from algae, fungiand unicellular organisms.

The washing solvents, extracting solvents, or recovery solvents, in thecontext of the present invention these terms are interchangeable, caninclude but are not limited to the lower alcohols such as methanol,ethanol, propanol or isopropanol; ketones, such as acetone; water, and acombination of the lower alcohol, or ketone, with water.

A person skilled in the art of extraction of naturally-occurring plantconstituents will recognize that a number of different extractionsmethods exist in the literature, including percolation, vat extraction,counter-current extraction, etc. The particular method of extractionused is not important to the process of the present invention.

The present invention uses hydrophobic interaction chromatography aloneor in combination with other separation techniques to isolate thecompound of interest. In this respect, the invention defined in thepresent application can be combined with the separation techniquesdefined in Applicant's co-pending application entitled “A Process forthe Purification of Non-Polar Extractives”, which usesaliphatic-substituted polysaccharide gel matrices in a process ofhydrophobic interaction chromatography.

The process of purifying the non-polar extractive, according to thepresent invention involves three basic steps: absorption, washing andrecovery. According to one aspect of the present invention, the processinvolves a fourth optional step of regenerating the column, without theuse of a harsh chemical treatment or the generation of excessive salt ornon-recoverable processing waste stream.

According to the present invention, separation and purification of awide range of extractives with similar solubilities in aqueous alcoholicsolvents can be effected based on their differential binding tospecifically-modified polysaccharide gels. Such extractives can include:steroids and triterpenoids, such as saponins, cardiac glycosides andsteryl conjugates; flavonoids, such as flavones, flavonols, isoflavonesand all of their glycosides; phenolic conjugates, such as aliphaticalcohol esters and amides; polar lipids, such as mono- and di-glyceridesand their derivatives and alk(en)ylresorcinols; and prolamines, such aszein, avenin, hordein or gliadin.

In establishing whether a compound is considered to be bound to the geland to have exhibited hydrophobic interaction with the gel, thefollowing criteria must be met:

-   -   a) it must be soluble in, or form a micelle or stable emulsion        in, the solvent with which it is loaded onto the column, and        with which the column is washed; and    -   b) it must be retained by the gel after washing with at least        1.25×V_(b) of the washing solvent, wherein V_(b) is the packed        bed volume of the column; wherein the washing solvent is a lower        alcohol in combination with water in a ratio sufficient to        retain said compound.        This latter criterion must be met since porous gels of the types        described herein show molecular size exclusion capabilities.        These effects are not however observed beyond approximately        1.25×V_(b) and therefore are not involved in the processes or        practices described.

In the recovery step, the proportion of the organic solvent in theelution aqueous organic solvent is increased to decrease the hydrophobicbinding of the extractive to the gel and thus elute the extractive. Insome embodiments of the present invention the extractive can be elutedwith the initial washing stream.

In the present invention, reference will be made to the degree ofrelative hydrophobic interaction of specific compounds or groups and/orclasses of compounds by the use of a dimensionless constant, K′ definedas the ratio of the number of mL of a particular solvent to move thecompounds through a volume of 1 mL of gravity packed gel. Since thisdimensionless constant is independent of column dimensions (i.e. length,diameter, etc.), the conditions described herein can be used for scaledup operations over several magnitudes.

As noted previously, an optional fourth step of the present invention isthe regeneration of the column, without the use of any harsh chemicalsor the generation of excessive salt or non-recoverable processing wastestream. Since conditions for each application have been establishedwherein the compounds of interest have been totally removed from thegel, the column can be regenerated and re-equilibrated in the startingsolvent. Surprisingly, it has been found that a simple washing of thegels with a suitable solvent such as 95% ethanol or isopropanol issufficient in most cases to remove any material appearing to be adheringto the gels at the end of a process application. The gels are thenre-equilibrated with starting solvent for immediate reuse. In thismanner, regeneration and recycling up to at least 4000 times over 10years have been observed without noticeable loss of effectiveness in theprocesses described herein. Clean-in-place/sanitation procedures wheredeemed appropriate can be effected using dilute NaOH as permanufacturers recommendations (Amersham Pharmacia Biotech manuals,technical bulletins, etc.).

The purification of the compound, according to the present invention,can be carried out at any suitable temperature, known to persons skilledin the art. The column separations can be accomplished at temperaturesranging from about 2° C. to 60° C. Temperature ranges from about 4° C.to 30° C., being more commonly used.

While this invention is described in detail with particular reference topreferred embodiments thereof, said embodiments are offered toillustrate but not to limit the invention.

EXAMPLES Example 1 Isolation of Oat Saponins from Oat Flour UsingHydrophobic Interaction Chromatography on Octyl Sepharose CL-4B™ andNovel Substituted Hydrophobic Gels

Two different types of saponins have been isolated from oat kernels: theavenacoside-type pentacyclic triterpenoid saponins and the avenacin-typesteroidal saponins. The structures of two avenacosides, avenacoside Aand avenacoside B have been elucidated (Tschesche, R., et al., Chem.Ber., 102, 2072-2082, 1969; Tschesche, R. and Lauven, P., Chem. Ber.,104, 3549-3555, 1971), but at least 2 other avenacosides remainuncharacterized (Kesselmeier, J. and Strack, D., Z. Naturforsch. 36C,1072-1074, 1981). Several of the avenacin-type saponins have also beenstructurally identified (Crombie, L., et al., J. Chem. Soc., Perk.Trans. 1, 1917-1922, 1986).

In the case of the avenacosides A and B, the steroid moiety isglycosylated with a total of 5 and 4 sugar residues respectively, whilethe 4 avenacins are the same trisaccharide derivative of 4 similar butnot identical sapogenols. The co-occurrence of such complex mixtures ofneutral saponins makes their separation difficult either as groups orindividual components, from extracts which contain sugars andoligosaccharides of similar composition. In the following example,extensive use was made of hydrophobic interaction chromatography tofirst allow group separation of the saponins and secondly, to facilitatethe purification of specific components of each type of saponin.

First, HIC on Octyl Sepharose CL-4B™ in aqueous 50% ethanol was utilizedto carry out a group separation of all the oat saponins of both types,amongst others, from polar and non-polar non-saponin components. Thenall the oat saponins were further purified as a group by removingcharged components from this saponin-containing fraction by cationexchange chromatography on SP Sephadex C-25™ in the hydrogen form anddouble anion exchange chromatography on QAE Sephadex A-25™ in theacetate and hydroxyl forms, as described below. Since the oat saponinsare uncharged, they will not be absorbed by any of these steps and willpass through all three types of columns along with other neutralcomponents. Finally, HIC on both Octyl Sepharose CL-4B™ and SP SephadexC-25™ in the hexadecyltrimethylammonium (HDTMA⁺) form were used toseparate the avenacin saponins from the avenacoside saponins and topurify individual members of these two groups. It should be emphasizedthat during the saponin purification processes described below, a numberof non-saponin fractions were also generated that were highly enrichedin other components. Since the methods described herein involved ethanoland water as the only solvents, and volatile acids and bases as the onlyreagents, these fractions represent excellent sources for additionalvalues.

Thus, a sample of Avena saliva L. cult. Tibor was milled to pass througha 1 mm screen and 25 g of the resulting oat flour added with vigorousstirring to 125 mL (solids/liquids=1/5) refluxing acidified aqueous 80%ethanol (ethanol:water:glacial acetic acid 80:19:1 v:v:v). The mixturewas heated for an additional 20 minutes with continuous stirring underreflux. After cooling to approximately 4° C., the resuspended mixturewas transferred to a volumetrically graduated glass column fitted with acoarse porosity fritted disk, and allowed to warm to room temperature(25° C.) and settle by gravity to give a packed bed of known volume(V_(b)). The column was then drained and washed with 2×V_(b) freshacidified aqueous 80% ethanol, and this percolation-type extractionprocess repeated twice. After draining, the extracted solid residue wasremoved from the percolation extraction column and air-dried to constantweight to determine the percentage of oat flour extracted. The extractsand washings were combined and were concentrated in vacuo to an oilysyrup by rotary evaporation at 40° C., and represented 16% by weight ofthe original oat groats. The oily syrup was then dispersed in aqueous80% ethanol, 5 mL of Octyl Sepharose CL-4B™ beads in aqueous 80% ethanoladded, and the mixture evaporated to a thick slurry in vacuo by rotaryevaporation at 40° C. The slurry was then resuspended in aqueous 50%ethanol and transferred to a graduated column of 45 mL Octyl SepharoseCL-4B™, pre-equilibrated and gravity packed in the aqueous 50% ethanolsolvent. The column (final V_(b) 50 mL) was then washed with 3×V_(b) ofthe same solvent, to give a washings fraction with K′≦3 containing allthe oat saponins, sugars, amino acids, and salts amongst others, and aK′≧3 fraction, still hydrophobically bound to the gel. This K′≧3fraction, containing carotenoid pigments, acyl glycerides, polar lipids,some of the oat prolamines and free sterols amongst others, wasrecovered and the gel recycled by first passing 2×V_(b) of aqueous 95%ethanol through the column to recover the bound material, and then2×V_(b) of re-equilibrating solvent.

Thus the K′≦3 fraction prepared above was first treated to removecomponents such as cationic prolamines, peptides, amino acids andinorganic cations, by chromatography on SP Sephadex C-25™ in thehydrogen form. Accordingly, a volumetrically graduated column containing50 mL (=V_(b); i.e. 2 mL gel/gm oat groats extracted) of SP SephadexC-25™ in the sodium form (as received from the manufacturer) was swollenin aqueous 50% ethanol and converted to the hydrogen form by treatingthe column, with a 3-fold milli-equivalent excess of 0.1N HCl in aqueous50% ethanol, and washing the column until the washings were pH ˜6. TheK′≦3 fraction prepared above and dissolved in 5 mL of aqueous 50%ethanol was then loaded onto the column and the column washed with3×V_(b) of fresh aqueous 50% ethanol. The washing (i.e. K′≦3 fraction)containing the oat saponins, amongst others, was evaporated to drynessin vacuo by rotary evaporation at 40° C. This K′≦3 fraction was thentreated to remove components such as anionic prolamines, peptides, andamino acids, organic acids and inorganic anions, all of which carry anet negative charge at or below pH 6. Accordingly, a 50 mL (=V_(b); i.e2 mL gel/gm oat groats extracted) QAE Sephadex A-25™ anion exchangecolumn in the chloride form (as received from the manufacturer) wasswollen in aqueous 50% ethanol and first converted to the hydroxyl formby treating the column with a 3-fold milliequivalent excess of 1.0N NaOHin aqueous 50% ethanol and washing the column until the washings were pH˜8. The column was then converted to the acetate form by treating thecolumn with a 3-fold milliequivalent excess of 1% (v:v) glacial aceticacid in aqueous 50% ethanol and washing the column with aqueous 50%ethanol until the washings were pH ˜6. The saponin-containing fractionin 5 mL of aqueous 50% ethanol was then loaded onto the column and thecolumn washed with 3×V_(b) of fresh aqueous 50% ethanol. The washing(i.e. K′≦3 fraction) containing the oat saponins, amongst others, wasevaporated to dryness in vacuo by rotary evaporation at 40° C. Finally,to remove the flavonoids, phenolics and other non-saponins carrying anet negative charge at pH ˜8, the saponin fraction was passed through a50 mL (=V_(b); i.e 2 mL gel/gm oat groats extracted) QAE Sephadex A-25™column anion exchange column in the hydroxyl form, prepared as describedabove. The saponin-containing fraction in 5 mL of aqueous 50% ethanolwas then loaded onto the column and the column washed with 3×V_(b) offresh aqueous 50% ethanol. The washings (i.e. K′≦3 fraction) containingprimarily the oat saponins, galactoglycerides, neutral amino acids andsugars were concentrated in vacuo by rotary evaporation at 40° C. Thepreliminary fractionation scheme is summarized in FIG. 1.

Preliminary TLC examination of this K′≦3 fraction, as described below,showed the presence of a number of different coloured spots with thep-anisaldehyde detecting reagent including brown (free sugars), green(avenacoside-type sapogenins), grayish-blue (avenacin-type saponins),purple (galactosylglycerides) and yellow(lysophosphatides).

Hydrophobic interaction chromatography on Octyl Sepharose CL-4B™ and SPSephadex C-25™ in the hexadecyltrimethylammonium (HDTMA⁺) form were alsoused to separate the non-saponin components, to separate the avenacinsaponins from the avenacoside saponins and to isolate individual membersof these two groups from each other employing the following series ofprotocols. First the non-saponins and minor sapogenin mono- anddi-glycosides were separated from the avenacins and avenacosides onOctyl Sepharose CL-4B™. The individual avenacins were then separated byfurther chromatography on Octyl Sepharose CL-4B™, and individual membersof the more polar avenacosides, on SP Sephadex C-25™ in the HDTMA⁺form.However, in order to isolate and identify individual components of theoat saponin mixture, an additional oat sample (100 g) was processed byscaling up the above procedure using the same column types, solvents andproportions. In this way a further approximately 300 mg of freeze-driedsaponins were produced in a single run.

Thus, an aliquot (266.5 mg) of the freeze-dried saponins was dissolvedin 10 mL acidified aqueous 40% ethanol (i.e. ethanol:water:glacialacetic acid 40:59.9:0.1 v:v:v). The solution was absorbed onto a 100 mLcolumn (=V_(b); 2.66 mg saponin/mL gel) of Octyl Sepharose CL-4B™,equilibrated and gravity packed in the same solvent. The column waswashed with 2×V_(b) of fresh acidified aqueous 40% ethanol to give aK′≦2 sub-fraction which was evaporated to dryness in vacuo by rotaryevaporation at 40° C. The column was then eluted with 2×V_(b) of aqueous80% ethanol, to recover absorbed components and this sub-fraction (i.e.K′≧2 sub-fraction) similarly evaporated to dryness in vacuo by rotaryevaporation at 40° C. TLC examination of the K′≦2 sub-fraction showedseveral blue fluorescent spots under long wave UV (365 nm) typical ofthe avenacins containing the N-methylanthraniloyl moiety, at least 4green spots typical of the avenacosides when sprayed with thep-anisaldehyde reagent, and a large zone of high R_(f) corresponding tothe sugars and neutral amino acids amongst others. The K′≧2 sub-fractionby TLC examination contained a blue fluorescent spot at the origin underlong wave UV (365 nm) typical of the aglycone of theN-methylanthraniloyl-containing avenacin sapogenol and both purple andyellowish spots, characteristic of certain classes of lipids whensprayed with p-anisaldehyde reagent as noted above. Thus, this firststep effected the separation of both avenacin-type saponins andavenacoside-type saponins, recovered in the K′≦2 sub-fraction, fromnon-saponins (i.e. K′≧2 sub-fraction).

The next step involved the separation of the avenacin-type saponins fromthe avenacoside-type saponins and other remaining non-saponincomponents, by taking advantage of lower degree of glycosylation of theavenacins relative to the avenacosides and therefore their greaterhydrophobic binding potential. Accordingly, the K′≦2 sub-fraction wasdissolved in 10 mL acidified aqueous 20% ethanol (i.e.ethanol:water:glacial acetic acid 20:79.9:0.1 v:v:v) and absorbed onto a100 mL column (=V_(b)) of Octyl Sepharose CL-4B™, equilibrated andgravity packed in the same solvent. The column was washed with 2×V_(b)of fresh acidified aqueous 20% ethanol to give a K′≦2 sub-fraction whichwas evaporated to dryness in vacuo by rotary evaporation at 40° C.Components still absorbed on the column were then recovered using2×V_(b) of aqueous 80% ethanol, and this sub-fraction (i.e. K′≧2sub-fraction) similarly evaporated to dryness in vacuo by rotaryevaporation at 40° C. TLC examination of the 2 sub-fractions revealedthat the K′≦2 sub-fraction contained the avenacosides along with thefree sugars and neutral amino acids amongst others, but no detectableavenacins, while the K′≧2 sub-fraction contained the avenacins (i.e. the“avenacin sub-fraction’) along with traces of low R_(f) avenacosides.

Finally, to remove the neutral amino acids and sugars from theavenacosides, the K′≦2 sub-fraction was chromatographed on a gravitypacked 25 mL (=V_(b); i.e 1 mL gel/gm oat groats extracted) SP SephadexC-25™ in the hexadecyltrimethylammonium (HDTMA⁺) form, prepared asdescribed above. Thus, the K′≦3 fraction in 5 mL of aqueous 25% ethanolwas absorbed onto the column and washed with 3×V_(b) of fresh aqueous25% ethanol to give a K′≦3 sub-fraction devoid of saponins (TLC). Thesaponins absorbed on the column were then recovered with 3×V_(b) ofaqueous 80% ethanol, evaporated to dryness in vacuo by rotaryevaporation at 40° C., and freeze-dried to an off-white powder (i.e. the“avenacoside sub-fraction”; yield: 77.5 mg; 0.31% dry basis). The flowdiagram for the separation and recovery of the 2 major oat saponin typesis summarized in FIG. 2.

Thin Layer Chromatography (TLC)

TLC of saponins was performed on MKC₁₈F reverse phase plates (1×3 in.,200 μm thickness, Whatman International Ltd, Maidstone, UK) using thesolvent system methanol:aqueous 5% acetic acid (75:25 v:v)(i.e.methanol:water:glacial acetic acid 75:23.5:1.5 v:v:v). Compounds werevisualized by spraying with a 0.5% solution (v:v) of p-anisaldehyde inacidified aqueous ethanol (i.e. ethanol:concentrated sulfuricacid:water:p-anisaldehyde 90:5:4.5:0.5 v:v:v:v) and heating at 100° C.for 3 min. This reagent gives a number of distinct colors with differentconstituents including brown (free sugars), transitory yellow quicklyturning to pink, green, or grayish-blue (saponins), slowly appearingreddish (amino acids, prolamines), purple (galactosylglycerides), andyellow (lysophosphatides).

High Performance Liquid Chromatography (HPLC)

HPLC analyses of saponins were conducted using a Thermo SeparationsProducts solvent delivery system and data collecting software (PC 1000)on a C₁₈ CSC Hypersil column (250×4.6 mm, 120 Å, 5 μm). A “massdetector” (evaporative light scattering detector, ELSD, Alltech VarexMKII), with the drift tube temperature set at 120° C. and the gas flowat 3.06 SLPM was used to detect all compounds present in the injectionsample. The solvent system consisted of acetonitrile, water and aqueous5% acetic acid as shown below:

Time Acetonitrile Water 0 20 80 25 40 60 30 100 0 35 100 0 40 20 80

The flow rate was 1.0 mL/min.

Mass Spectrometry (MS)

Tandem liquid chromatography-mass spectrometry (HPLC-MS) analyses ofpure compounds were performed using flow injection (FIA) with no column.The mobile phase consisted of methanol:water (70:30 v:v) and the flowrate was 1001 L/min. Solvents were delivered using a Hewlett Packard1100 binary pump. Mass spectrometry analyses were conducted using aMicromass Quattro Spectrometer with an upgraded hexapole sourceoperating in the electrospray positive mode. Scanning was done in therange of from 200 to 1500 m/z units with a cone voltage of 100 or 200volts.

The “avenacin sub-fraction” prepared above was subjected to HPLC-ELSDanalysis and a typical HPLC profile is shown in FIG. 3 a. Thissub-fraction corresponds to the 4 avenacins of Crombie et al. (Crombie,L., et al., J. Chem. Soc., Perk. Trans. 1, 1917-1922, 1986.). The“avenacoside sub-fraction” HPLC-ELSD profile (FIG. 3 b), on the otherhand, contained 5 peaks with 2 major peaks (Peaks 3 and 4). Of these,Peaks 3 and 4 had similar retention times to those reported foravenacosides B and A respectively, as reported by Kesselmeier and Strack(Kesselmeier, J. And Strack, D., Z. Naturforsch. 36C, 1072-1074, 1981).

It was found that individual components of both the avenacin-typesaponins (i.e avenacins A-1, A-2 etc.) and the avenacoside-type saponins(i.e avenacosides A, B, C etc.) could also be separated by hydrophobicinteraction chromatography by the judicious choice of both gel type andwashing solvent composition. Thus, the avenacins-type saponins, whichare relatively more non-polar than the avenacosides, were fractionatedand purified on Octyl Sepharose CL-4B™ using isocratic washing withaqueous 25% and 30% ethanol. The avenacosides, containing more sugarresidues than the avenacins, were fractionated and purified on SPSephadex C-25™ in the HDTMA⁺ form, since these avenacosides were notcompletely retained on Octyl Sepharose CL-4B™, even with water as thesolvent.

For example, the “avenacin sub-fraction” prepared above was taken up in10 mL aqueous 30% ethanol and absorbed onto a 30 mL (=V_(b)) graduatedcolumn of Octyl Sepharose CL-4B™ gravity packed and equilibrated in thesame solvent. The column was then washed with the same solvent andfractions corresponding to the following K′ values collected andanalyzed for avenacins by TLC and HPLC. The fraction with K′ from 2.5 to5.5 contained all of the major avenacins observed in the HPLC profile(Peak 3, FIG. 3 a) along with small amounts of minor avenacins andtraces of avenacoside-like compounds. Further chromatography of thisfraction on the same column but using aqueous 25% ethanol yielded themajor avenacin peak in the fraction with K′ from 6 to 8 at greater than80% purity as determined by UV spectrophotometric (diode-array detectionat 365 nm) and HPLC-ELSD analyses. Mass spectral analyses of thisisolated peak confirmed its identity as avenacin A-1. FIG. 4 a shows anHPLC-ELSD profile of the fraction obtained from the HIC column, whileFIG. 4 b shows the mass spectrum obtained from this peak, and thestructure assigned to the avenacin is illustrated in FIG. 4 c.Similarly, the remaining peaks can be individually purified simply byvarying the amount of ethanol and water in the isocratic solvent toeither increase or decrease the relative hydrophobic binding of thecomponents in the mixture.

In a further example, the “avenacoside sub-fraction” prepared above wastaken up in 10 mL aqueous 32.5% ethanol and absorbed onto a 30 mL(=V_(b)) graduated column of SP Sephadex C-25™ in the HDTMA⁺form,gravity packed and equilibrated in the same solvent. The column was thenwashed with the same solvent and fractions with K′<2.5, K′ 2.5 to 5 andK′>5 collected and analyzed for avenacosides by TLC and HPLC. Thefraction with K′<2.5 contained primarily avenacoside Peaks 2 and 3; K′2.5 to 5 contained avenacoside Peaks 1 and 3 with small amounts of Peak4, and the K′>5 fraction contained primarily avenacoside Peaks 4 and 5with a small amount of Peak 3.

Example 2 Isolation of Saponins from Quinoa Flour Using HydrophobicInteraction Chromatography on Octyl Sepharose CL-4B™ and NovelSubstituted Hydrophobic Gels

In the following example the quinoa saponins were isolated and highlypurified as an entire group using HIC. Advantage was also taken of thefact that none of the saponins carries a cationic charge within theworking range of the invention, and thus passage of the extractedmixture through a cation exchange column will not remove any of thesaponins from the mixture. A final saponin-enriched fractionsubstantially free of interfering compounds of like solubility wasprepared using only ethanol, water, and volatile acids and bases.

Thin Layer Chromatography (TLC)

TLC was performed as described in Example 1.

High Performance Liquid Chromatography (HPLC)

HPLC analyses were conducted using the same equipment as described inExample 1 except a C₈ Keystone column 120 Å, 5 μm, (250×4.6 mm) was usedand a UV diode array detector (Thermo Separation Products, SpectraSYSTEMUV 3000), monitoring at 270 nm (general phenolic functional groupabsorption) was linked in tandem with the ELSD detector operated as inExample 1. This allowed simultaneous detection of non-saponin phenoliccomponents. The solvent system consisted of acetonitrile, H₂O, andaqueous 5% glacial acetic acid (v %):

Time Acetonitrile H₂O 5% Acetic acid 0 15 75 10 25 25 65 10 29 100 0 033 100 0 0 35 15 75 10The flow rate was 1.0 mL/min.

Thus, quinoa seeds (Cultivar Colorado 407 (CO07)) were ground to pass 20mesh to produce a flour. To a heated solution (60° C.) of 100 mL ofacidified aqueous 80% ethanol (ethanol:water:glacial acetic acid80:19:1, v:v:v), 25 g of the flour was carefully added with stirring(solids/liquids=1/4 v:v). The mixture was allowed to stir for 30 minutesand then cooled to room temperature. The mixture was centrifuged(2830×g, 7 minutes) and the supernatant drawn off. The pellet was washedwith fresh solvent (200 ml) and re-centrifuged. The supernatant wasdrawn off and the pellet re-suspended a third time with fresh solvent.All supernatants were combined and filtered through a course sinteredglass filter. The dark yellow filtrate, constituting an extract, had apH of 7.03 and contained 7.3% solubles as determined by gravimetricanalysis of a freeze-dried sub-sample.

The extract was evaporated to dryness in vacuo by rotary evaporation at40° C. and taken up in 2.5 ml of acidified aqueous 50% ethanol(ethanol:water:glacial acetic acid 50:49:1, v:v:v). The cloudysuspension was loaded onto a graduated glass column of Octyl SepharoseCL-4B™ column (final packed V_(b)=25 mL; i.e. 1 mL gel/gm quinoa flourextracted) pre-equilibrated and gravity packed in acidified aqueous 40%ethanol (ethanol:water:glacial acetic acid 40:59:1, v:v:v). The columnwas then washed with 2×V_(b) of the acidified aqueous 50% ethanol togive a K′≦2 fraction which was concentrated in vacuo at 40° C. by rotaryevaporation to a yellow syrup. TLC analyses of this fraction (K′≦2)showed it contained the quinoa saponins, sugars, amino acids, organicacids, and some of the pigments amongst others. The material remainingon the column was removed (and the column regenerated for re-use), bypassing 2×V_(b) of acidified aqueous 80% ethanol to give a K′≧2fraction. TLC of this fraction showed it contained the bulk of the polarlipids and some of the pigments but no traces of saponin. Gravimetricanalyses showed that this K′≧2 fraction constituted about 1.2% of theoriginal quinoa flour while the saponin-enriched K′≦2 fractionrepresented over 6.4% of the flour.

The K′≦2 fraction prepared above was taken up in 50 mL of aqueous 50%ethanol and applied to a graduated glass column of SP Sephadex C-25™ inthe ammonium form (final packed V_(b)=25 mL; i.e. 1 mL gel/gm quinoaextracted), pre-equilibrated and gravity packed in aqueous 50% ethanol.The column was then washed with 2×V_(b) of the aqueous 50% ethanol togive a K′≦2 fraction, containing the neutral and anionic components,which was concentrated in vacuo at 40° C. by rotary evaporation to apale yellow syrup. The cationic material on the column was thenrecovered by passing 2×V_(b) of 5% ammoniacal aqueous 50% ethanol (i.e.ethanol:water:conc. ammonium hydroxide 50:45:5 v:v:v) and byconcentrating this K′≧2 fraction in vacuo at 40° C. by rotaryevaporation to remove the solvent and excess ammonia. By gravimetricanalyses, this cationic fraction was found to represent about 1% of thequinoa flour while the K′≦2 fraction constituted more than 5.3%. TLCanalyses of these fractions showed that all the saponins were present inthe K′≦2 fraction along with the sugars, organic acids, and some of theamino acids, protein and pigments amongst others, while the K′≧2fraction showed no traces of saponins.

Further purification of the saponins was carried out using HIC on a 25mL graduated glass column of SP Sephadex C-25™ in the HDTMA⁺ form,prepared as in Example 1 (final packed V_(b)=25 mL; i.e. 1 mL gel/gmquinoa extracted), pre-equilibrated and gravity packed in aqueous 20%ethanol. The anionic and neutral K′≦2 fraction prepared above was takenup in 2.5 mL aqueous 20% ethanol, loaded onto the column and washed with2×V_(b) of the 20% aqueous ethanol to give a K′≦2 fraction. The materialstill absorbed on the column was then recovered by passing 2×V_(b) ofaqueous 80% ethanol through the column to give an enriched saponinfraction. Both fractions were then evaporated to dryness in vacuo at 40°C. by rotary evaporation and analyzed by gravimetric analysis, TLC andHPLC. The K′≦2 fraction representing about 4.75% of the original quinoaflour was devoid of saponins while the K′≧2 fraction constituting about0.59%, showed a highly enriched saponin content by both HPLC and TLC.The scheme for the isolation and group separation of quinoa saponins issummarized in FIG. 5.

HPLC-ELSD with simultaneous HPLC-UV analyses of this enriched quinoasaponin fraction showed that the fraction w as substantially free of major interfering non-saponin components. The HPLC-ELSD profile showed 3major peaks at retention times of between 21 and 29 minutes. Whencompared on the same scale of response, the HPLC-UV profile showedseveral minor non-saponin peaks containing phenolic functions absorbingat 270 nm, eluting between 4 and 8 minutes, that were also present inthe ELSD profile. TLC analysis showed these non-saponin components toinclude phenolic pigments, some amino acids, and some protein-likematerial, amongst others. Fractional integration of all the ELSD peaksenabled the approximate purity of this enriched saponin fraction to beestimated at greater than 90%.

Considering the elution gradient used in the HPLC analysis, the quinoasaponins can be roughly divided into two major groups, as shown in FIGS.6 a and 6 b, based on relative hydrophobicity. The relatively lesshydrophobic quinoa Group 1 saponins eluted from between 14 and 27minutes during that portion of the gradient utilizing from 15% to 25%acetonitrile in the solvent. This group consisted of at least 12chromatographically distinct saponins with peaks 5 and 6 const tulingthe major components. Quinoa Group 2 saponins on the other hand elutedonly after rapid ramping of the elution gradient to 100% acetonitrileand contained one major component, peak 13, and at least 2 additionalsaponins, bringing the total to at least 15 saponins from the particularcultivar.

As a further example of the chromatographic purification applications ofthe present invention, the quinoa saponins were subjected to additionalfractionation into Group 1 saponins and Group 2 saponins, and theremaining non-saponin components simultaneously removed. In thisapplication, use was made of the differences between the two saponingroups and their relative hydrophobic binding to a novel substitutedhydrophobic gel. Some of the non-saponin components were first removedby anionic exchange chromatography.

Thus, the enriched saponin fraction prepared above (i.e. K′≧2 in 20%ethanol on SP Sephadex C-25™ in the HDTMA⁺form) was evaporated todryness under reduced pressure and taken up in aqueous 50% ethanol (5ml). The yellowish solution was applied to a 25 mL anion exchange columnof QAE Sephadex A-25™ prepared essentially as described in Example 1except converted to the formate form in stead of the acetate form, andpre-equilibrated in aqueous 50% ethanol (final packed volume V_(b)=25ml; i.e. 1 ml gel/gm quinoa flour extracted). The column was then washedwith 2×V_(b) of aqueous 50% ethanol to give a K′≦2 fraction which wasevaporated to dryness in vacuo at 40° C. by rotary evaporation. Thematerial still absorbed onto the column was then removed by passing3×V_(b) of acidified aqueous 50% ethanol (ethanol:water:formic aceticacid 50:45:5 v:v:v) through the column to produce a K′≧2 faction whichwas also evaporated to dryness in vacuo at 40° C. by rotary evaporation.Both fractions were then analyzed by TLC and HPLC. As shown in thereproduction of the TLC plate of the K′≦2 fraction (FIG. 7 a), thisfraction contained all of the saponins along with the yellow pigments.No saponins were detected in the K′≧2 fraction.

The next step was to separate the quinoa Group 1 saponins from Group 2saponins by HIC on SP Sephadex C-25™ in the HDTMA⁺ form. Thus the K′≦2fraction from the previous anion exchange step was taken up in 2.5 mL ofaqueous 50% ethanol and absorbed onto a graduated glass column of SPSephadex C-25™ in the HDTMA⁺ form, prepared and gravity packed aspreviously described and pre-equilibrated in aqueous 50% ethanol (finalpacked volume V_(b)=25 ml; i.e. 1 mL gel/gm quinoa flour extracted). Thecolumn was then washed with 2×V_(b) of aqueous 50% ethanol to give aK′≦2 fraction. The material still absorbed on the column was thenremoved by passing 2×V_(b) of aqueous 80% ethanol through the column toproduce a K′≧2 fraction. Both fractions were then evaporated to drynessin vacuo at 40° C. by rotary evaporation and analyzed by TLC and HPLC.As shown in the reproduction of the TLC plate of the K′≦2 fraction (FIG.7 b), this fraction contained essentially only the Group 1 quinoasaponins as determined by HPLC-ELSD analysis and was devoid of pigments.By comparison of HPLC-ELSD fractional integration and the TLC spotintensities, the major zone on the TLC plate with R_(f) values between0.19 and 0.26 (FIG. 7 b) corresponded to peak 13. Fractional integrationof all the ELSD peaks enabled the approximate purity of this saponinfraction to be estimated at greater than 99%. The K′≧2 fractioncontained the rest of the saponins and the phenolic pigments which couldbe separated by a final treatment with the same HIC gel but using asolvent with a higher water content. Thus, the K′≧2 fraction containingthe Group 2 saponins and the pigments was taken up in 2.5 mL of aqueous35% ethanol and absorbed onto the same column of SP Sephadex C-25™ inthe HDTMA⁺form, pre-equilibrated in aqueous 35% ethanol (final packedvolume V_(b)=25 ml; i.e. 1 mL gel/gm quinoa flour extracted). The columnwas washed with 4×V_(b) of aqueous 35% ethanol to give a K′≦4 fractionwhich was evaporated to dryness in vacuo at 40° C. by rotary evaporationto give a yellowish lacquer. The material still absorbed on the columnwas then removed by passing 2×V_(b) of aqueous 60% ethanol through thecolumn to produce a K′≧4 fraction. Rotary evaporation of this fractionto dryness in vacuo at 40° C. gave a white powder. TLC and HPLC-ELSDanalyses of the K′≦4 revealed no saponins in this fraction but most ifnot all of the pigments. On the other hand, as shown in the reproductionof the TLC plate of the K′≧4 fraction (FIG. 7 c), this fractioncontained essentially only the Group 2 quinoa saponins as determined byHPLC-ELSD analysis and was devoid of pigments. The major peaks 5 and 6appeared as close running zones on the TLC plate with R_(f) valuesbetween 0.40 and 0.48. Fractional integration of all the ELSD peaksenabled the approximate purity of this saponin fraction to be estimatedat greater than 99%. The scheme for the purification and separation ofquinoa Group 1 and 2 saponins is summarized in FIG. 8.

Example 3 Comparative Utility of Ionically-Substituted vs.Covalently-Substituted Gels for the Separation and Purification ofSaponins using Hydrophobic Interaction Chromatography

Macroporous chromatographic media, based on a polysaccharide backboneand containing any one of a number of different covalently-linked alkylligands with chain lengths up to 12 carbons, are commercially available.Examples of such products include for example Butyl Sepharose™ AmershamPharmacia Biotech, Piscataway, N.J.), aminohexyl agarose(C₆) and dodecylagarose(C₁₂) (Sigma Chemical Co., St. Louis, Mo.). These gels, for themost part suffer from low ligand substitution rates, typically ≦40 μMligand/mL gel, resulting in relatively low hydrophobic interactioncapacity for low molecular weight compounds, and high costs. Accordingto the present invention, hydrophobic interaction chromatographicseparations can be effectively and efficiently carried out onmacroporous, highly substituted anionically- or cationically-chargedpolysaccharide gels using any one of a wide variety of hydrophobiccounterions. Such gels are easily and reversibly generated at the timeof use and are stable under most physiological and biochemical workingconditions.

The practice of reverse phase and hydrophobic interaction chromatographyteaches that, in general, under identical elution conditions therelative binding affinity of a specific substrate to a hydrophobicligand substituted on the stationary phase will increase with increasingchain length of the ligand. To show the utility of thesereadily-modified gels and to compare their relative binding properties,a series of experiments was performed using identical column andsubstrate conditions but varying the type of stationary phase gel ligandchain length and the recovery solvent composition (i.e. aqueousethanol). The gel matrices were as follows: Butyl Sepharose™ and OctylSepharose CL-4B™ as received from the manufacturer; QAE Sephadex A-25™in the dodecyl sulfate form, prepared as described below, and SPSephadex C-25™ in the hexadecyltrimethylammonium form, prepared asdescribed below. It should be pointed out that none of these gelmatrices show any appreciable absorption or fluorescence in long wave UVlight (365 nm). The substrate was the saponin avenacin A-1, prepared andpurified from oat groats as described in Example 1. This saponincontains an N-methylanthraniloyl moiety which is stronglyautofluorescent in solution at 365 nm permitting on-column monitoring ofits migration through the gel matrix simply by observing the graduatedglass column under long wave UV light. Column beds were 30 mLgravity-packed in a volumetrically-graduated glass column fitted with amedium-porosity polypropylene fritted disk.

Basically, either of the two different types of ion exchangers, anionicor cationic, preferably consisting of an ionically-substitutedpolysaccharide gel, can be used as the hydrophilic core. To this core ahydrophobic ligand, containing a strongly ionizable functional group ofopposite charge to that of the polysaccharide, is attached by ionexchange so that all, or substantially all, of the available ionic sitesin the original polysaccharide are occupied by the ligand to form amodified, hydrophobic phase component. Thus, in this example QAESephadex A-25™ anion exchange chromatography gel, in the chloride form(as received from the manufacturer), containing 0.5 milliequivalents/mLexchange capacity (Amersham Pharmacia Biotech, Piscataway, N.J.) wasswollen and equilibrated in aqueous 50% ethanol and gravity packed intoa volumetrically-calibrated chromatography column to give a bed of knownvolume. The column was first converted to the hydroxide form by passingexcess 0.5N NaOH in 50% ethanol through the column and washing the bedto neutral pH with aqueous 50% ethanol. The bed was then converted tothe lauryl sulfate form by passing a 2-fold milliequivalent excess ofsodium dodecyl sulfate in aqueous 50% ethanol (SDS, Fisher Scientific,Ottawa, ON) and again washing the column with aqueous 50% ethanol toremove any excess SDS. By using a strongly ionized (e.g. sulfate)anionic detergent containing a linear aliphatic component of 12-carbonsthe chromatographic media has been substantially modified to give a HICcolumn which is relatively stable over the pH range 2.5-11 and hasincreased capacity for hydrophobic interaction due to the longer alkylchain length, than readily available gels (C-12 vs. C-8 forOctyl-Sepharose CL-4B™), and about 10 times greater degree ofsubstitution (for example, nominally 0.5 millimoles/mL vs. 50 μmoles/mLfor Octyl-Sepharose CL-4B™), while still maintaining attributes suitablefor HIC applications.

Similarly, a modified chromatography media suitable for HIC was preparedusing an analogous process and a cationic ion exchanger. Thus, forexample, SP Sephadex C-25™ cation exchange chromatography gel, in thesodium form (as received from the manufacturer), containing 0.3milliequivalents/mL exchange capacity (Amersham Pharmacia Biotech,Piscataway, N.J.) was swollen and equilibrated in aqueous 50% ethanoland gravity packed into a volumetrically-calibrated chromatographycolumn to give a bed of known volume. The column was first converted tothe hydronium form by passing an excess of 0.1N HCl in aqueous 50%ethanol through the column and washing the bed to neutral pH withaqueous 50% ethanol. The bed was then converted to thehexadecyltrimethylammonium form by passing a 2-fold milliequivalentexcess of hexadecyltrimethylammonium bromide in aqueous 50% ethanol(HDTMA, Sigma Chemical Co., St. Louis, Mo.) and washing the column withaqueous 50% ethanol to remove any excess HDTMA. Again, by using astrongly ionized (e.g. quaternary amine) cationic detergent containing alinear aliphatic component of 16-carbons the chromatographic media hasbeen substantially modified to give a HIC column, which is relativelystable over the pH range 2.0-13, and has increased capacity forhydrophobic interaction due to the longer alkyl chain length, thanreadily available gels (C-16 vs. C-8 for Octyl Sepharose CL-4B™), andalmost 10 times greater degree of substitution (for example, nominally0.3 millimoles/mL vs. 50 μmoles/mL for Octyl Sepharose CL-4B™), whilestill maintaining attributes suitable for HIC applications.

The degree of relative hydrophobic interaction of the substrate wasobtained from K′ values, as previously defined by direct measurement ofthe on-column fluorescent band. K′ values were recorded for both theleading and tailing edge of the fluorescent zone and these K′ valuesaveraged. The results are summarized in Table 1 using SP Sephadex C-25™columns in the alkyl trimethylammonium form substituted with differentchain lengths.

TABLE 1 Effect of Hydrophobic Ligand Chain Length on Binding of AvenacinA-1 Washing Solvent (EtOH:H₂O) Average K′ Value with Different LigandSide Chains (v:v) C₄ C₈ C₁₂ C₁₄ C₁₆ C₁₈ 10:90 12.75 19.5 20:80 5.6611.75 30:70 1.74 3.99 40:60 1.24 1.45 4.83 5.25 7.56 9.82 50:50 1.191.63 1.68 1.92 2.44 60:40 1.08 1.18 1.26 1.55

These results are depicted in FIGS. 9 and 10. In FIG. 9 the effect ofethanol concentration on K′ values of avenacin A-1 using different chainlength substitutions is shown. According to the present invention,separation will be most effective in the steep slope of the graph. Inthis area of the graph a small change in ethanol concentration resultsin a large change in K′ value, thus allowing effective separationbetween two compounds so that they can be isolated from each other. Onthe other hand, at the end of the graph, where the slopes of the curvesconverge, two compounds will elute from the column together, as changingthe ethanol concentration does not result in a large change in K′values. FIG. 10 is a plot of the same results showing the effect ofethanol concentration and substrate hydrophobicity on K′ values foravenacin A-1, as a function of the chain length. Thus these graphs canbe used to assist in choosing the optimum ethanol concentration andcolumn chain length for separations of compounds from, in this example,avenacin A-1. As will be clear to persons of ordinary skill in the art,similar series of experiments can be conducted on other compounds ofinterest to be isolated, in order to deduce an appropriate purificationstrategy.

Table 2 shows a comparison between the use of a QAE-Sephadex A-25™dodecyl sulfate column and a SP Sephadex C-25™ dodecyltrimethylammoniumcolumn. Although there are differences in the K′ values between the twocolumns, the relationship between K′ value and the concentration ofethanol is the same.

TABLE 2 Effect Column Types on Binding of Avenacin A-1 Average K′ ValueWashing QAE Sephadex SP Sephadex Solvent A-25 ™ Dodecyl C-25 ™ Dodecyl-(EtOH:H₂O) sulphate trimethylammonium (v:v) (C12) (C12) 10:90 20:8030:70 40:60 3.33 4.83 50:50 0.70 1.63 60:40 0.53 1.08

All scientific publications and patent documents are incorporated hereinby reference.

The present invention has been described with regard to preferredembodiments. However, it will be understood to persons skilled in theart that a number of variations and modifications can be made withoutdeparting from the scope of the invention as described in the followingclaims.

1. A method of isolating a non-polar extractive comprising: contactingsaid non-polar extractive in an aqueous organic solution with a gelmatrix selected from the group of an electrostatically-linked, aliphaticor alicyclic-substituted anionic or cationic polysaccharide gel; washingsaid gel matrix with said aqueous organic solvent solution; washing saidgel matrix with additional aqueous organic solvent solution, wherein theproportion of the organic solvent in said solution is increased; andrecovering said extractive from an effluent stream.
 2. The method ofclaim 1, further comprising regenerating the gel matrix for re-use. 3.The method according to claim 1, wherein the non-polar plant extractivesare selected from the group consisting of steroids and triterpenoids,flavonoids, phenolic conjugates, polar lipids, and prolamines.
 4. Themethod according to claim 3, wherein the steroids and triterpenoids areselected from die group consisting of saponins, cardiac glycosides andsteryl conjugates.
 5. The method according to claim 3, wherein theflavonoids are selected from the group consisting of flavones,flavonols, isoflavones and all of their glycosides.
 6. The methodaccording to claim 3, wherein the phenolic conjugates are selected fromthe group consisting of aliphatic alcohol esters and amides.
 7. Themethod according to claim 3, wherein the polar lipids are selected fromthe group consisting of mono- and di-glycerides and their derivates andalk(en)yl resorcinols.
 8. The method according to claim 3, wherein theprolamines are selected from the group consisting of zein, avenin,hordein and gliadin.
 9. The method according to claim 3, wherein thenon-polar extractives are from either synthetic or natural source. 10.The method according to claim 9, wherein the natural source of thenon-polar extractives is selected from the group consisting of plantmaterial, algae, fungi and unicellular organisms.
 11. The methodaccording to claim 10, wherein the plant material is selected from thegroup consisting of agricultural, viniculture, horticulture, aquacultureand plants native to lands and oceans.
 12. The method according to claim11, wherein the agricultural plant material is selected from the groupconsisting of wheat, oats, rye, corn, rice, quinoa, amaranth, buckwheat,triticale or barley; or oilseeds, such as soybean, canola, flaxseed,sunflower, safflower or mustard; or pulsecrops, for example, peas,lentils or beans; or forage crops, such as fescue, timothy, clover,alfalfa or wheatgrass; or herbs, such as parsley, rosemary, sage, ormint.
 13. The method according to claim 1, wherein the organic solventsolution is a solution containing a lower alcohol, a ketone or acombination thereof.
 14. The method according to claim 13, wherein thelower alcohol is selected from the group consisting of methanol,ethanol, propanol and isopropanol.
 15. The method according to claim 13,wherein the ketone is acetone.
 16. The method according to claim 1,wherein the cationic polysaccharide gel is a neutral polysaccharide ofthe anyhydrogalactan or dextran class containing covalently-linkedcationically-charged functional groups.
 17. The method according toclaim 16, wherein the cationically-charged functional groups areselected from the group consisting of tertiary amines and quaternaryamines.
 18. The method according to claim 1, wherein the cationicpolysaccharide gel is selected from the group consisting ofdiethyaminoethyl polydextran, quaternary aminoethyl polydextran, andquaternary amine crosslinked agarose.
 19. The method according to claim1, wherein the anionic polysaccharide gel is a neutral polysaccharide ofthe anyhydrogalactan or dextran class containing covalently-linkedanionically-charged functional groups.
 20. The method according to claim19, wherein the anionically-charged functional groups are selected fromthe group consisting of alk(en)yl sulfonates and alk(en)yl phosphonates.21. The method according to claim 1, wherein the anionic polysaccharidegel is selected from the group consisting of sulphonate crosslinkedagarose.
 22. The method according to claim 1, wherein the hydrophobicligand is an anionic detergent selected from the group consisting ofalk(en)yl sulfonates and sulfates, alk(en)ylbenzenesulfonates,taurocholates and taurodeoxycholates, alk(en)yl phosphonates andphosphates, and mono- and di-alk(en)ylphosphatidic acids.
 23. The methodaccording to claim 22, wherein the alk(en)yl substitution contains fromabout 4 to about 18 carbons.
 24. The method according to claim 22,wherein the anionic detergent is sodium dodecyl sulfate.
 25. The methodaccording to claim 1, wherein the hydrophobic ligand is a cationicdetergent selected from the group consisting ofalk(en)yltrimethylammonium halides and quaternary alk(en)ylammoniumhalides, quaternary alk(en)ylpyridinium halides, and alk(en)ylmagnesiumhalides.
 26. The method according to claim 25, wherein the alk(en)ylsubstitution contains from about 4 to about 18 carbons.
 27. The methodaccording to claim 25, wherein the cationic detergent ishexadecyltrimethylammonium bromide.